Gustin M C, Goodman D B
J Biol Chem. 1981 Oct 25;256(20):10651-6.
The mechanisms of ion movement across the apical membrane of the colon have previously been investigated only in intact tissue. To investigate these mechanisms directly, we have undertaken the isolation and characterization of the apical brush-border membrane of the rabbit descending colon. The purification protocol consists of an initial isolation of single epithelial cells after dissociation of the mucosal layer in EDTA, a high pH (8.3), low ionic strength homogenization of the cells, and differential centrifugation and separation of apical membrane from nuclei, and filamentous material on a 7.5% Percoll gradient. A 20-fold enrichment in alkaline phosphatase (an apical membrane enzyme marker) specific activity over the initial homogenate value is observed in the final membrane fraction. This fraction also contains a K+-activated, pH 7.8, optimum ATPase (20 times purified over homogenate) with the following properties: 1) low Kact (2 X 10(-4) M) for K+; 2) resistance to high ionic strength (1 M Tris) solubilization; 3) competitive inhibition by Na+ (K1 = 14 mM), no activation by Na+; 4) inhibition by orthovanadate (K1 = 40 nM), but no effect of oligomycin (20 micrograms/ml of protein) or ouabain (10(-3) M); and 5) a K+-sensitive phosphorylated intermediate. These characteristics suggest that this membrane-bound ATPase is distinct from other known ATPases including the Na+ + K+ - ATPase-Na+ pump of the basolateral membrane.
离子跨结肠顶端膜移动的机制此前仅在完整组织中进行过研究。为了直接研究这些机制，我们对兔降结肠的顶端刷状缘膜进行了分离和特性鉴定。纯化方案包括：首先在乙二胺四乙酸（EDTA）中解离粘膜层后分离单个上皮细胞，然后对细胞进行高pH值（8.3）、低离子强度的匀浆，接着通过差速离心从细胞核和丝状物质中分离顶端膜，并在7.5%的 Percoll 梯度上进行分离。在最终的膜组分中，碱性磷酸酶（一种顶端膜酶标记物）的比活性相对于初始匀浆值提高了20倍。该组分还含有一种钾激活的、pH值为7.8的最佳ATP酶（相对于匀浆纯化了20倍），具有以下特性：1）对钾的低激活浓度（2×10⁻⁴ M）；2）对高离子强度（1 M Tris）溶解具有抗性；3）受钠离子竞争性抑制（K₁ = 14 mM），不受钠离子激活；4）受原钒酸盐抑制（K₁ = 40 nM），但不受寡霉素（20微克/毫升蛋白质）或哇巴因（10⁻³ M）影响；5）存在一个对钾敏感的磷酸化中间体。这些特性表明，这种膜结合ATP酶与其他已知的ATP酶不同，包括基底外侧膜的钠钾ATP酶 - 钠泵。
